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Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids.

Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids. IIA .Z able to reduce Light-Mediated Conversion of Nitrogen logue significantly single- Dioxide to DNA strand breaks in Chinese hamster Nitric Oxide by Carotenoids fibroblasts to The lung exposed NO2. pro- tection afforded by may be y-tocopherol Robert V. Cooney, Patricia J. Harwood, Laurie J. Custer, and related to its to reduce to the ability NO2 A. Adrian Franke more stable NO molecule without requir- University of Hawaii, Cancer Research Center of Hawaii, HI Honolulu, 96813 USA Nitrosation of ing light (22). primary amines on DNA bases in vivo can result (e.g., nitroprusside) are converted, upon in directly genotoxic damage; therefore, light exposure, to nitrosating species, such formation of a nitrite ester, capable of as NO' or nitrogen oxide radicals nitrosating amines, could limit protection (14-16), which can subsequently react with nucle- from nitrosative damage associated with ophiles to form nitroso compounds NO2 exposure. Conversion of NO2 to (14,1X. NO may additionally protect against Exposure of plants to nitrogen dioxide oxidative of damage by way the relative in the dark causes acute injury and accu- antioxidant of NO To properties (23). mulation of nitrite, whereas simultaneous provide into the insight mechanism of exposure to light reduces toxicity and dark-phase NO2 toxicity, we used an in nitrite formation (18,19). The mechanism vitro model to system study the reactions of NO2 toxicity in plants has not been elu- of carotenoids with NO2 and the effects of cidated, but involves both direct light on the products obtained. likely effects from exposure to this highly reactive As shown in Figure 1, fl-carotene (10- free radical and indirect effects from the M in reacted with hexane) NO2 to pro- products formed when NO2 reacts with duce NO in the presence of light, whereas cellular constituents. Although exposure to control solutions in the dark showed no nitrogen oxides affects photosynthesis (20), significant NO evolution. Measured NO it is not known if this is related to the levels in the gas phase exiting n-carotene acute of toxicity NO2. Shimazaki et al. solutions began to decrease significantly (18) have proposed that the accumulation after 6-8 min of exposure to NO2, corre- of nitrite in the dark and subsequent light- sponding to the observed decolorization of mediated reaction of nitrite with the chloro- carotenoid solution (Fig. 2) and Carotenoids are phyll produced by plants to pro- accounts for the observed toxicity of destruction of the highly conjugated poly- tect against oxidative and photolytic dam- NO2. Such an explanation, however, does ene system (color loss occurred indepen- age (1). Considerable indirect not provide a evidence mechanistic basis to fully dently of light indicating that the initial suggests that dietary antioxidants may also explain either the differential accumulation reaction between NO2 and n-carotene was provide protective functions in longer-lived of nitrite in dark not versus light-exposed light mediated). Higher concentra- animal species, including humans (2,3). plants or the manner by which simultane- tions of n-carotene (10-4 M) showed sus- Although the nature of oxidants in ous to reduces acting exposure light toxicity. tained NO emission during the 15-min mammalian in systems may some respects The high reactivity of carotenoids, exposure and were not completely decol- differ from those in the found in plants, underlying many green plants, toward oxi- orized over this time (data not shown). mechanisms be In may similar. elucidating dants and free radicals suggests that Although 8 min of exposure to in NO2 the the mechanisms) of NO2 as well carotenoids an toxicity, may play important role in dark resulted in complete loss of color and as its is mitigation by antioxidants, it essen- preventing free radical damage caused by the disappearance of all n-carotene as mea- tial to consider both the direct NO2 and indirect exposure. However, the reaction sured by HPLC, 24 hr later samples toxicities manifested both by NO2 and its products of highly conjugated regained some color as well as small molecules, a reaction products. such as the carotenoids, with NO2 have amount of in samples exposed P-carotene The highly reactive NO2 radical is not been well less than 10 minutes the characterized; consequently, (Fig. 2). Lutein, formed from the oxidation of the conditions under nitric oxide which carotenoids major carotenoid found in dark green, which is (NO), produced in high-tempera- protect against NO2 damage in plants or leafy vegetables such as also spinach, ture combustion processes, by bacteria animals is unknown. or Reaction of NO2 reduced NO2 to NO in the light (Fig. 3), normal through biochemical processes in with double such as those found in as did an bonds, lycopene, acyclic carotenoid both plants and animals (4-6). Nitrogen linoleic acid, or cholesterol, results in the found in and a tomatoes, canthaxanthin, dioxide damages living cells in a variety of formation of food colorant two keto nitrosating agents, which containing groups ways, including oxidation of membrane have been identified as nitrite esters (21). (data not shown). lipids (7, N-nitrosamine formation (8), like cholesterol and Carotenoid solutions to Alpha-tocopherol, exposed NO2 direct mutation of DNA DNA linoleic reacts with to form as evidenced (9-11), sin- acid, NO2 a formed a nitrosating agent by breaks and/or gle-strand (12), other oxida- nitrosating agent, while lack- y-tocopherol, tive reactions. Nitrogen dioxide accumula- a in ing methyl group the ortho position to Address to R.V. correspondence Cooney, tion in elevators is University of Cancer Research Center of grain associated with the phenol, does not Gamma-toco- Hawaii, (22). 1236 Lauhala HI Hawaii, Street, Honolulu, 96813 "silo filler's disease" which is is also more effective at characterized pherol preventing USA. massive and often lethal by lung damage in neoplastic transformation of cultured cells This research was supported by grant ES-04302 exposed individuals The free radical a that be related to its (13). (22), property may from the National Institute of Environmental reactions of occur NO2 generally indepen- unique with oxides. reactivity nitrogen Health Sciences and the Leahi Trust. of while more stable Bittrich et al. that dently light, species Recently (12) reported Received 13 August 1993; accepted 23 February such as nitrite and metal 1994. nitrosyl complexes 'y-tocopherol was the only tocopherol ana- Environmental Health Perspectives A * : ; . *i ; e *. E their ability to subsequently nitrosate mor- pholine (Fig. 4). Beta-carotene samples exposed to light during NO2 treatment _ i S ss| w | , i, B _ uz nesswB showed significantly less ability to nitrosate CL E BEIM 4I + B II morpholine = (54% decrease, p 0.006 for the difference in as well as means) less 0. 0.~~~~~~~~~~~~~~~~~R Griess-Saltzmann reactive material (42% decrease, p = less 0.008), indicating nitrite and/or nitrite ester present. Lutein accu- mulated less nitrite/nitrite ester relative to under both lighting conditions P-carotene and showed a reduction in corresponding o!!1P0 1 1 4 16 the ability to nitrosate morpholine (Fig. 4). 2 4 6 12 1 lime (min) Other carotenoids examined showed a sim- Time (mim) ilar pattern of nitrosation and light-medi- 1. Effect of on NO formation Figure light by NO2- 3. NO formation lutein and in Figure the by ated inhibition in of their NO2 spite structural carotene. Beta-carotene M in 5 ml exposed (1O of Lutein 5 (i0 M in 5 ml presence light. hexane) differences, although the ability of lyco- hexane) was for the indicated times to exposed was for the indicated times to 27.5 exposed ppm to in the 27.5 in at a flow rate of pene exposed NO2 light to subse- ppm by bubbling NO2 N2 in at a flow rate of 60 mI/min by bubbling NO2 N2 60 mI/mmn. NO was measured in the exit quently nitrosate morpholine was more gas by and NO measured in the exit as described in gas direct of 10O-gI into a ther- injection gas samples variable for undetermined reasons 1. this were (data Figure main- During period samples mal as described energy analyzer previously (22). tained either in the dark or illuminated a halo- not shown). by 1 _ I were in total darkness Samples kept during expo- -0N Each the mean of three gen light. point represents __U_ _ll Carotenoids are lipophilic compounds sure to or 10 cm from a placed quartz-halo- NO2 determinations ±SE. separate that act as membrane antioxidants in plants 60-watt with an neutral gen lamp intervening to filter to block infrared and protect against free radical and oxidative density light prevent Each the mean sample warming. point represents damage (1). The double bonds in carote- of three determinations ± SE. separate noids make these compounds highly reactive 20~~~~~~~ toward singlet oxygen (25) or free radicals such as The NO2 (11). tocopherols also pre- vent oxidative damage by reacting with radi- cals to form less reactive intermediates (26). E Interestingly, y-tocopherol is found primari- ly in seed oils, whereas chloroplasts contain - 7 a-tocopherol and carotenoids (27). Whether this differential localization in plant tissues is related to conditions of light and/or NO2 o 5 exposure is unknown; however, it is consis- tent with the observed superiority of y-toco- to pherol reduce NO2 to NO in the dark (22). The light-mediated conversion of NO2 to NO by plant antioxidants offers a plau- Figure 4. Nitrosation of morpholine and nitrite accumulation by carotenoids exposed to NO2 in sible mechanism to explain decreased sensi- Time (min) hexane. Nitrite accumulation and nitrosating tivity to NO2 in toxicity plants exposed to light. In addition to reducing the level of Figure 2. Change in A450 for carotene solutions potential of carotenoid or control solutions P-ca exposed to NO2. Samples of irotene (10 M in exposed to NO2 for 15 min in either the dark or nitrite and/or nitrite esters formed and P-ca light as described in Figure 1 were measured. hexane), exposed as described = n Figure 1 3) subsequent nitrosation reactions, the pro- (n were measured immediately a fter at After NO exposure was stopped, the volume exposure duction of nitric oxide may provide addi- the indicated times for absorb)ance at 450 nm was readjusted to 5 ml with hexane and an tional antioxidant protection through its (A450). Samples that were remeaisured 24 hr later aliquot removed for nitrite analysis (24). Five min- reaction with NO and other intracellular showed some recovery of A450, iridicatingthatthe utes later, 50 pl of 100 mM morpholine and N- free radicals to block radical chain pro- loss was in part reversible. Contrrol solutions of - nitrosopyrolidine (200 pg/ml as internal standard) carotene in hexane bubbled ml/mmn) with N2 in hexane was added to the 5-ml sample. One reactions. (60 pogation The reaction of NO milliliter of this solution was then into an plotted ,n = placed in the dark or are alsi only light 1)2 with NO2 to form N203 might prevent HPLC analysis indicated a srnnall quantity of amber vial and incubated at 370C. A 1-pI sample (22) , acute free radical damage from NO2, carotene at 24 hr versiis none immedi- was removed after 24 hr and analyzed by gas present although increasing delayed damage arising chromatography-thermal energy analysis for N- ately after exposure to NO2. nitrosomorpholine as described previously (22). from the nitrosation of amines by N203 Data represent the mean of three separate while the reaction of NO with lipid or with olefins to form N NO2 O and the cor- experiments ± SE. radicals would result in for -IC X~n oxygen chain ter- responding epoxide. The rmation or ilu mination. Such a mechanism may explain and an as to nitrite ester particularly with regard to the epoxide opposed effect of the of formation superior ability to pre- depends on the nature of the light and neighboring substituents on the y-tocopherol vent single-strand DNA breaks in cells olefinic as well as the reaction nature of formed. compound products Temperature exposed to NO2 (12). also a conditions employed. Recerntly Bors et al. may play role, particularly in ani- Insight into the chemical mechanism (29) have shown that oxida and mals, as we have observed increased rates Ltive geno- which or by y-tocopherol illuminated toxic damage from 2-nitrop ropane in vivo of NO2 reduction to NO at by tocopherols carotenoids reduce NO2 to NO is provid- is mediated through NO2 formation and not 370C (data shown). ed in a recent Bosch the direct paper by and Kochi reaction of NC with DNA Although the role of carotenoids in )2 (28) in which they describe the reaction of bases. Further work in this area is antioxidant needed, providing protection in plants Volume Number 1994 102, 5, May 461 FAr &jfflf;-. .l is well accepted, an analogous function for 7. Pryor WA, Lightsey 20. Hill AC, Bennett JH. Inhibition of apparent JW. Mechanisms of nitrogen dioxide photosynthesis by nitrogen oxides. Atmos in humans has not reactions: initiation of lipid dietary antioxidants peroxidation and the production of nitrous Environ 4:341-348(1970). been conclusively established, despite acid. Science 21. Mirvish SS, Babcock DM, Deshpande AD, 214:435-437(1981). numerous epidemiologic studies that show 8. Challis Shucker Nagel DL. Identification of cholesterol as a BC, DEG, Fine DH, Goff an inverse association between carotenoid EU, Hoffman GA. Amine nitration and nitro- mouse skin lipid that reacts with nitrogen can- consumption and/or serum levels and sation by aqueous nitrogen dioxide. In: N- dioxide to yield a nitrosating agent, and of cer incidence (3,30). Cigarette smoke, nitroso compounds: occurrence and biological cholesteryl nitrite as the nitrosating agent pro- effects (Bartsch H, O'Neill IK, Castegnaro M, duced in a chemical system from cholesterol. which contains large quantities of nitrogen Okada M, eds), IARC Scientific Publication Cancer Lett 31:97-104(1986). oxides (31), is strongly associated with no. 22. 41. Lyon:International Agency for Cooney RV, Franke AA, Harwood PJ, Hatch- lung cancer incidence in humans, and Research on Cancer, 1982;1 1-20. Pigott V, Custer Mordan LU. y-Toco- Li, many studies suggest a protective effect for 9. Wink DA, Kasprzak KS, Maragos CM, pherol detoxification of nitrogen dioxide: antioxidants against lung cancer (32). Elespuru RK, Misra M, Dunams TM, Cebula superiority to a-tocopherol. Proc Natl Acad Interestingly, a study of Fiji Islanders, who TA, Koch WH, Andrews AW, Allen JS, Sci USA 90:1771-1775(1993). Keefer LK. DNA Wink lung cancer incidence deaminating ability and 23. DA, Hanbauer I, Krishna MC, DeGraff have extremely low of nitric oxide and its Gamson Mitchell W, J, JB. Nitric oxide pro- genotoxicity progeni- rates, revealed only weak associations, neg- tors. Science 254:1001-1003(1991). tects against cellular damage and cytotoxicity atively with carotenoids and positively with 10. Nguyen Bruson D, Crespi CL, T, Penman from reactive oxygen species. Proc Natl Acad but did suggest a strong a-tocopherol, BW, Wishnok JS, Tannenbaum SR. DNA Sci USA 90:9813-9817(1993). with plasma inverse association y-toco- damage and mutation in human cells exposed 24. Saltzman BE. Colorimetric micro-determina- pherol (33). nitric to oxide in vitro. Proc Natl Acad Sci tion of nitrogen dioxide in the atmosphere. USA 89:3030-3034(1992). Anal Chem 26:1949-1954(1954). The observations described here offer a Arroyo PL, Hatch-Pigott V, Mower HF, 25. Di Mascio P, Kaiser S, Sies H. Lycopene as plausible explanation for the differential Cooney RV. Mutagenicity of nitric oxide and the most efficient biological carotenoid singlet toxicity of NO2 in plants exposed under its inhibition by antioxidants. Mutat Res oxygen quencher. Arch Biochem Biophys conditions and suggest various lighting 281:193-202(1992). 274:532-538(1989). limitations in the ability of carotenoids to 12. vitamin E in Bittrich H, Matzig AK, Kraker I, Appel KE. 26. Packer L. Protective role of bio- nitrogen oxide-mediated DNA Am Clin Nutr protect against N02-induced single strand breaks are logical systems. 53:1050S- inhibited by in antioxidative vitamins in V79 1055S(1991). damage in plants the absence of light. cells. vitamin E in Chem-Biol Interact 86:199-211(1993). 27. Draper HH. Role of plants, The conditions under which carotenoids 13. In: Lowry T, Schuman LM. "Silo-fillers dis- microbes, invertebrates and fish. Vitamin and tocopherols protect against oxidative ease" -a syndrome caused by nitrogen diox- E: a comprehensive treatise (Machlin ed). LJ, in as well as the nature of damage animals, ide. Am J Med Assoc 162:153-160(1956). New York:Marcel Dekker, 1980;391-395. the oxidative species, remain to be deter- Challis BC, Li BFL. Formation of N- 28. Bosch E, Kochi JK. Electron-transfer catalysis A of the dark- mined. better understanding nitrosamines and N-nitramines by photolysis. of olefin epoxidation with nitrogen dioxide Chem Soc Chem In: N-nitroso compounds: occurrence and (dinitrogen tetroxide). J phase reactions of plant antioxidants may biological effects (Bartsch H, O'Neill IK, Commun 667-668(1993). to elucidate their roles in ultimately help Stettmaier Castegnaro M, Okada M, eds), IARC 29. Bors W, Michel C, Dalke C, K, human health as well. intermediates Scientific Publication no. 41. Saran M, Andrae U. Radical of The Lyon:International Agency for Research on during the oxidation nitropropanes. REFERENCES its Cancer, 1982;31-39. formation of NO2 from 2-nitropropane, 15. Bonnett R, Charalambides AA, Martin RA, reactivity with nucleosides, and implications 1. Krinsky NI. Antioxidant functions of car- Chem Sales KD, Fitzsimmons, BW. Reactions of for the genotoxicity of 2-nitropropane. otenoids. Free Rad Biol Med 7:617-635 nitrous with acid and nitric oxide prophyrins Res Toxicol 6:302-309(1993). (1989). and cancer and Haems. Nitrosylhaems as nitrosating 30. Mathews-Roth, MM. Carotenoids 2. Cutler RG. Carotenoids and retinol: their agents. J Chem Soc Chem Commun 884- prevention-experimental and epidemiologi- possible importance in determining longevity 885(1975). cal studies. Pure Appl Chem 57:717-722 of primate species. Proc Natl Acad Sci USA 16. Butler AR, Glidewell C, Reglinski J, Waddon (1985). 81:7627-7631(1984). in A. The pentacyanonitrosylferrate ion. Part 1. 31. Norman V, Keith CH. Nitrogen oxides 3. Ziegler RG. A review of epidemiologic evi- Reactions with various amines. J Chem Res tobacco smoke. Nature 205:915-916(1965). dence that carotenoids reduce the risk of can- 5:279(1982). 32. Menkes MS, Comstock GW, Vuillemier JP, cer. J Nutr 119:1 16-122(1989). R. 17. Cooney RV, Ramseyer J, Ross PD. Amine Helsing KJ, Rider AA, Brookmeyer Serum 4. Goretski J, Hollocher TC. The kinetic and nitrosation by sodium nitroprusside. Cancer beta-carotene, vitamins A and E, selenium, isotopic competence of nitric oxide as an Lett 40:213-218(1988). and the risk of lung cancer. N Engl J Med intermediate in denitrification. J Biol Chem 18. Shimazaki K, Yu SW, Sakaki T, Tanaka K. 315:1250-1254(1986). 265:889-895(1990). Differences between spinach and kidney bean 33. Le Marchand L, Hankin JH, Bach F, 5. Klepper LA. Nitric oxide emissions from soy- in terms of to plants sensitivity fumigation Henderson BH, Kolonel LN. Cancer risk fac- in reductase bean leaves during vivo nitrate with NO2. Plant Cell tor in the Physiol 33:267-273 survey South Pacific. Presented at assays. Plant Physiol 85:96-99(1987). the XVII Pacific (1992). Science Congress, Honolulu, 6. Nathan C. Nitric oxide as a T, Sasakawa H. Transformation of secretary product 19. Yoneyama HI, 27 May-1 June 1991. of mammalian cells. FASEB J 6:3051-3062 absorbed in leaves. atmospheric NO2 spinach (1992). Plant Cell Physiol 20:263-266(1979). 462 Environmental Health Perspectives http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Health Perspectives Unpaywall

Light-mediated conversion of nitrogen dioxide to nitric oxide by carotenoids.

Cooney, R V; Harwood, P J; Custer, L J; Franke, A A
Environmental Health Perspectives – May 1, 1994
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Abstract

IIA .Z able to reduce Light-Mediated Conversion of Nitrogen logue significantly single- Dioxide to DNA strand breaks in Chinese hamster Nitric Oxide by Carotenoids fibroblasts to The lung exposed NO2. pro- tection afforded by may be y-tocopherol Robert V. Cooney, Patricia J. Harwood, Laurie J. Custer, and related to its to reduce to the ability NO2 A. Adrian Franke more stable NO molecule without requir- University of Hawaii, Cancer Research Center of Hawaii, HI Honolulu, 96813 USA Nitrosation of ing light (22). primary amines on DNA bases in vivo can result (e.g., nitroprusside) are converted, upon in directly genotoxic damage; therefore, light exposure, to nitrosating species, such formation of a nitrite ester, capable of as NO' or nitrogen oxide radicals nitrosating amines, could limit protection (14-16), which can subsequently react with nucle- from nitrosative damage associated with ophiles to form nitroso compounds NO2 exposure. Conversion of NO2 to (14,1X. NO may additionally protect against Exposure of plants to nitrogen dioxide oxidative of damage by way the relative in the dark causes acute injury and accu- antioxidant of NO To properties (23). mulation of nitrite, whereas simultaneous provide into the insight mechanism of exposure to light reduces toxicity and dark-phase NO2 toxicity, we used an in nitrite formation (18,19). The mechanism vitro model to system study the reactions of NO2 toxicity in plants has not been elu- of carotenoids with NO2 and the effects of cidated, but involves both direct light on the products obtained. likely effects from exposure to this highly reactive As shown in Figure 1, fl-carotene (10- free radical and indirect effects from the M in reacted with hexane) NO2 to pro- products formed when NO2 reacts with duce NO in the presence of light, whereas cellular constituents. Although exposure to control solutions in the dark showed no nitrogen oxides affects photosynthesis (20), significant NO evolution. Measured NO it is not known if this is related to the levels in the gas phase exiting n-carotene acute of toxicity NO2. Shimazaki et al. solutions began to decrease significantly (18) have proposed that the accumulation after 6-8 min of exposure to NO2, corre- of nitrite in the dark and subsequent light- sponding to the observed decolorization of mediated reaction of nitrite with the chloro- carotenoid solution (Fig. 2) and Carotenoids are phyll produced by plants to pro- accounts for the observed toxicity of destruction of the highly conjugated poly- tect against oxidative and photolytic dam- NO2. Such an explanation, however, does ene system (color loss occurred indepen- age (1). Considerable indirect not provide a evidence mechanistic basis to fully dently of light indicating that the initial suggests that dietary antioxidants may also explain either the differential accumulation reaction between NO2 and n-carotene was provide protective functions in longer-lived of nitrite in dark not versus light-exposed light mediated). Higher concentra- animal species, including humans (2,3). plants or the manner by which simultane- tions of n-carotene (10-4 M) showed sus- Although the nature of oxidants in ous to reduces acting exposure light toxicity. tained NO emission during the 15-min mammalian in systems may some respects The high reactivity of carotenoids, exposure and were not completely decol- differ from those in the found in plants, underlying many green plants, toward oxi- orized over this time (data not shown). mechanisms be In may similar. elucidating dants and free radicals suggests that Although 8 min of exposure to in NO2 the the mechanisms) of NO2 as well carotenoids an toxicity, may play important role in dark resulted in complete loss of color and as its is mitigation by antioxidants, it essen- preventing free radical damage caused by the disappearance of all n-carotene as mea- tial to consider both the direct NO2 and indirect exposure. However, the reaction sured by HPLC, 24 hr later samples toxicities manifested both by NO2 and its products of highly conjugated regained some color as well as small molecules, a reaction products. such as the carotenoids, with NO2 have amount of in samples exposed P-carotene The highly reactive NO2 radical is not been well less than 10 minutes the characterized; consequently, (Fig. 2). Lutein, formed from the oxidation of the conditions under nitric oxide which carotenoids major carotenoid found in dark green, which is (NO), produced in high-tempera- protect against NO2 damage in plants or leafy vegetables such as also spinach, ture combustion processes, by bacteria animals is unknown. or Reaction of NO2 reduced NO2 to NO in the light (Fig. 3), normal through biochemical processes in with double such as those found in as did an bonds, lycopene, acyclic carotenoid both plants and animals (4-6). Nitrogen linoleic acid, or cholesterol, results in the found in and a tomatoes, canthaxanthin, dioxide damages living cells in a variety of formation of food colorant two keto nitrosating agents, which containing groups ways, including oxidation of membrane have been identified as nitrite esters (21). (data not shown). lipids (7, N-nitrosamine formation (8), like cholesterol and Carotenoid solutions to Alpha-tocopherol, exposed NO2 direct mutation of DNA DNA linoleic reacts with to form as evidenced (9-11), sin- acid, NO2 a formed a nitrosating agent by breaks and/or gle-strand (12), other oxida- nitrosating agent, while lack- y-tocopherol, tive reactions. Nitrogen dioxide accumula- a in ing methyl group the ortho position to Address to R.V. correspondence Cooney, tion in elevators is University of Cancer Research Center of grain associated with the phenol, does not Gamma-toco- Hawaii, (22). 1236 Lauhala HI Hawaii, Street, Honolulu, 96813 "silo filler's disease" which is is also more effective at characterized pherol preventing USA. massive and often lethal by lung damage in neoplastic transformation of cultured cells This research was supported by grant ES-04302 exposed individuals The free radical a that be related to its (13). (22), property may from the National Institute of Environmental reactions of occur NO2 generally indepen- unique with oxides. reactivity nitrogen Health Sciences and the Leahi Trust. of while more stable Bittrich et al. that dently light, species Recently (12) reported Received 13 August 1993; accepted 23 February such as nitrite and metal 1994. nitrosyl complexes 'y-tocopherol was the only tocopherol ana- Environmental Health Perspectives A * : ; . *i ; e *. E their ability to subsequently nitrosate mor- pholine (Fig. 4). Beta-carotene samples exposed to light during NO2 treatment _ i S ss| w | , i, B _ uz nesswB showed significantly less ability to nitrosate CL E BEIM 4I + B II morpholine = (54% decrease, p 0.006 for the difference in as well as means) less 0. 0.~~~~~~~~~~~~~~~~~R Griess-Saltzmann reactive material (42% decrease, p = less 0.008), indicating nitrite and/or nitrite ester present. Lutein accu- mulated less nitrite/nitrite ester relative to under both lighting conditions P-carotene and showed a reduction in corresponding o!!1P0 1 1 4 16 the ability to nitrosate morpholine (Fig. 4). 2 4 6 12 1 lime (min) Other carotenoids examined showed a sim- Time (mim) ilar pattern of nitrosation and light-medi- 1. Effect of on NO formation Figure light by NO2- 3. NO formation lutein and in Figure the by ated inhibition in of their NO2 spite structural carotene. Beta-carotene M in 5 ml exposed (1O of Lutein 5 (i0 M in 5 ml presence light. hexane) differences, although the ability of lyco- hexane) was for the indicated times to exposed was for the indicated times to 27.5 exposed ppm to in the 27.5 in at a flow rate of pene exposed NO2 light to subse- ppm by bubbling NO2 N2 in at a flow rate of 60 mI/min by bubbling NO2 N2 60 mI/mmn. NO was measured in the exit quently nitrosate morpholine was more gas by and NO measured in the exit as described in gas direct of 10O-gI into a ther- injection gas samples variable for undetermined reasons 1. this were (data Figure main- During period samples mal as described energy analyzer previously (22). tained either in the dark or illuminated a halo- not shown). by 1 _ I were in total darkness Samples kept during expo- -0N Each the mean of three gen light. point represents __U_ _ll Carotenoids are lipophilic compounds sure to or 10 cm from a placed quartz-halo- NO2 determinations ±SE. separate that act as membrane antioxidants in plants 60-watt with an neutral gen lamp intervening to filter to block infrared and protect against free radical and oxidative density light prevent Each the mean sample warming. point represents damage (1). The double bonds in carote- of three determinations ± SE. separate noids make these compounds highly reactive 20~~~~~~~ toward singlet oxygen (25) or free radicals such as The NO2 (11). tocopherols also pre- vent oxidative damage by reacting with radi- cals to form less reactive intermediates (26). E Interestingly, y-tocopherol is found primari- ly in seed oils, whereas chloroplasts contain - 7 a-tocopherol and carotenoids (27). Whether this differential localization in plant tissues is related to conditions of light and/or NO2 o 5 exposure is unknown; however, it is consis- tent with the observed superiority of y-toco- to pherol reduce NO2 to NO in the dark (22). The light-mediated conversion of NO2 to NO by plant antioxidants offers a plau- Figure 4. Nitrosation of morpholine and nitrite accumulation by carotenoids exposed to NO2 in sible mechanism to explain decreased sensi- Time (min) hexane. Nitrite accumulation and nitrosating tivity to NO2 in toxicity plants exposed to light. In addition to reducing the level of Figure 2. Change in A450 for carotene solutions potential of carotenoid or control solutions P-ca exposed to NO2. Samples of irotene (10 M in exposed to NO2 for 15 min in either the dark or nitrite and/or nitrite esters formed and P-ca light as described in Figure 1 were measured. hexane), exposed as described = n Figure 1 3) subsequent nitrosation reactions, the pro- (n were measured immediately a fter at After NO exposure was stopped, the volume exposure duction of nitric oxide may provide addi- the indicated times for absorb)ance at 450 nm was readjusted to 5 ml with hexane and an tional antioxidant protection through its (A450). Samples that were remeaisured 24 hr later aliquot removed for nitrite analysis (24). Five min- reaction with NO and other intracellular showed some recovery of A450, iridicatingthatthe utes later, 50 pl of 100 mM morpholine and N- free radicals to block radical chain pro- loss was in part reversible. Contrrol solutions of - nitrosopyrolidine (200 pg/ml as internal standard) carotene in hexane bubbled ml/mmn) with N2 in hexane was added to the 5-ml sample. One reactions. (60 pogation The reaction of NO milliliter of this solution was then into an plotted ,n = placed in the dark or are alsi only light 1)2 with NO2 to form N203 might prevent HPLC analysis indicated a srnnall quantity of amber vial and incubated at 370C. A 1-pI sample (22) , acute free radical damage from NO2, carotene at 24 hr versiis none immedi- was removed after 24 hr and analyzed by gas present although increasing delayed damage arising chromatography-thermal energy analysis for N- ately after exposure to NO2. nitrosomorpholine as described previously (22). from the nitrosation of amines by N203 Data represent the mean of three separate while the reaction of NO with lipid or with olefins to form N NO2 O and the cor- experiments ± SE. radicals would result in for -IC X~n oxygen chain ter- responding epoxide. The rmation or ilu mination. Such a mechanism may explain and an as to nitrite ester particularly with regard to the epoxide opposed effect of the of formation superior ability to pre- depends on the nature of the light and neighboring substituents on the y-tocopherol vent single-strand DNA breaks in cells olefinic as well as the reaction nature of formed. compound products Temperature exposed to NO2 (12). also a conditions employed. Recerntly Bors et al. may play role, particularly in ani- Insight into the chemical mechanism (29) have shown that oxida and mals, as we have observed increased rates Ltive geno- which or by y-tocopherol illuminated toxic damage from 2-nitrop ropane in vivo of NO2 reduction to NO at by tocopherols carotenoids reduce NO2 to NO is provid- is mediated through NO2 formation and not 370C (data shown). ed in a recent Bosch the direct paper by and Kochi reaction of NC with DNA Although the role of carotenoids in )2 (28) in which they describe the reaction of bases. Further work in this area is antioxidant needed, providing protection in plants Volume Number 1994 102, 5, May 461 FAr &jfflf;-. .l is well accepted, an analogous function for 7. Pryor WA, Lightsey 20. Hill AC, Bennett JH. Inhibition of apparent JW. Mechanisms of nitrogen dioxide photosynthesis by nitrogen oxides. Atmos in humans has not reactions: initiation of lipid dietary antioxidants peroxidation and the production of nitrous Environ 4:341-348(1970). been conclusively established, despite acid. Science 21. Mirvish SS, Babcock DM, Deshpande AD, 214:435-437(1981). numerous epidemiologic studies that show 8. Challis Shucker Nagel DL. Identification of cholesterol as a BC, DEG, Fine DH, Goff an inverse association between carotenoid EU, Hoffman GA. Amine nitration and nitro- mouse skin lipid that reacts with nitrogen can- consumption and/or serum levels and sation by aqueous nitrogen dioxide. In: N- dioxide to yield a nitrosating agent, and of cer incidence (3,30). 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DA, Hanbauer I, Krishna MC, DeGraff have extremely low of nitric oxide and its Gamson Mitchell W, J, JB. Nitric oxide pro- genotoxicity progeni- rates, revealed only weak associations, neg- tors. Science 254:1001-1003(1991). tects against cellular damage and cytotoxicity atively with carotenoids and positively with 10. Nguyen Bruson D, Crespi CL, T, Penman from reactive oxygen species. Proc Natl Acad but did suggest a strong a-tocopherol, BW, Wishnok JS, Tannenbaum SR. DNA Sci USA 90:9813-9817(1993). with plasma inverse association y-toco- damage and mutation in human cells exposed 24. Saltzman BE. Colorimetric micro-determina- pherol (33). nitric to oxide in vitro. Proc Natl Acad Sci tion of nitrogen dioxide in the atmosphere. USA 89:3030-3034(1992). Anal Chem 26:1949-1954(1954). The observations described here offer a Arroyo PL, Hatch-Pigott V, Mower HF, 25. Di Mascio P, Kaiser S, Sies H. Lycopene as plausible explanation for the differential Cooney RV. 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